Postprandial mammalian glucose utilization is regulated by insulin and the actions of this hormone are to suppress hepatic glucose output and stimulate glucose uptake into fat and muscle. For these tissues, insulin-dependent glucose uptake is achieved by the movement (translocation) of a vesicular pool of glucose transporters from an intracellular storage site to the cell surface, and it is the Glut4 transporter isoform that functions in this regard. Indeed the insulin sensitive transporter pool is a product of tissue specific gene expression, and the failure of insulin to mediate normal Glut4 translocation causes insulin resistance and type 2 diabetes. Moreover, the basic process of insulin signaling to vesicular traffic is a paradigm in cell biology the details of which remain incompletely described. Thus, insulin-dependent Glut4 translocation is of substantial basic and medical interest. We propose 3 specific aims to understand this process. The first aim is use specific antibodies to Glut4 and to other trafficking proteins to isolate Glut4 storage vesicles (GSVs) and identify their protein content. This information will point the way to more functional studies of individual GSV components such as over expression and gene knockdown/knockout experiments. The second Aim will use a known major cargo protein of GSVs, the insulin responsive aminopeptidase, as an affinity reagent to identify the cytoplasmic machinery, those adaptor and cytoskeletal proteins whose function mediates the specific protein sorting and vesicle trafficking of GSVs. Again the levels of such proteins will be raised and lowered to better understand their physiological functions. The third and last aim is based on our recent findings that APPL1, an adaptor protein involved in membrane trafficking and signaling, is a regulator of Akt action, Akt being the last kinase in the insulin signaling cascade. Thus we will characterize how APPL1 modulates Akt signaling in terms of APPL1 domains that interact with Akt and trafficking molecules such as Rab5. We hypothesize that there are multiple sites of Akt action that mediate Glut4 translocation, and we propose assays and strategies to define these sites. These studies will lead to a better understanding of pathways leading to clearance of glucose from the blood of direct relevance to type 2 diabetes mellitus. PUBLIC HEALTH RELEVANCE: The proposed research is to describe at the cellular and molecular level how insulin clears glucose from the blood. It is therefore directly relevant to type 2 diabetes because one or more molecular steps at the cellular level are impaired in this disease. Knowing what these steps are will point the way to better treatment of the disease.